BrdU labeling can be performed in vitro for cell lines and primary cell cultures, or in vivo for labeling cells within a living animal. During the BrdU assay, BrdU is incorporated into replicating DNA and can be detected using anti-BrdU antibodies.

After BrdU labeling, an additional DNA hydrolysis step (sometimes referred to as a DNA denaturing step) may be required after fixation and permeabilization to allow the anti-BrdU antibody access to the BrdU within the DNA.

We recommend using our ab6326 BrdU antibody. This product has been cited in hundreds of publications and independently reviewed by our customers with an average 5-star rating. The clone and its conjugates are manufactured in-house.

BrdU antibodies can be used in conjunction with cell type markers such as doublecortin, and NeuN to identify proliferating cells and newly differentiated neurons.

For rapid, convenient results, we also recommend our BrdU staining IHC kit which contains all the reagents you need for staining cells or tissues which have incorporated BrdU. Or consider a BrdU ELISA kit for a quantitative measure of BrdU incorporation, without imaging.

EdU staining is an alternative method with a shorter, simpler protocol than that required for BrdU.

Remove the existing culture medium from the cells and replace with 10 µM labeling solution.

Incubate the cells in the BrdU labeling solution for 1–24 hours at 37ºC in a CO2 incubator.

Note: BrdU incubation time depends on how rapidly the cells divide. Primary cells may need up to 24 hours, while rapidly proliferating cell lines may only need one hour. The exact time required to achieve the optimal signal-to-noise ratio should be optimized.

Remove the BrdU labeling solution from the cells and wash twice in PBS for about 5 seconds per wash.

Note: BrdU incorporation into rapidly dividing tissues, such as the small intestine, will be detectable as soon as 30 minutes post-injection. However, for most tissue, you may need to wait up to 24 hours. The exact treatment time and dosage will need to be optimized for your tissue of interest.

Oral administration

Oral administration of BrdU is a non-invasive procedure and therefore useful for extended BrdU administration, although it may introduce variability into experiments due to lack of control over an animal’s water consumption.

After treatment with BrdU, the animals can then be sacrificed according to standard protocols.

Fix and process tissue according to standard IHC protocols. However, before immunostaining refer to the DNA hydrolysis step below.

Note: for mice, 225 mg/kg per day of BrdU (calculated by measuring water consumption volumes per animal) should achieve sufficient BrdU labeling. However, the exact dose should be optimized for individual experimental conditions.

DNA hydrolysis

Incubate cells in 1–2.5 M HCL for 10 minutes to 1 hour at room temperature. The exact HCl concentration and incubation time should be optimized for your experiment. If using a shorter incubation time, incubating at 37oC may be more effective than room temperature.

Incubate sections in 1–2 M HCL for 30 minutes to 1 hour. The exact HCl concentration and incubation time should be optimized for your experiment. If using a shorter incubation time, incubating at 37oC may be more effective than room temperature.

Note: some researchers have reported that they don’t perform the HCl hydrolysis step and simply perform heat-induced epitope retrieval before continuing with immunostaining.

Co-staining with anti-BrdU

BrdU can be used in conjunction with other antibodies to identify proliferating and newly differentiated neurons. See below for our suggestions.

Ki67A cellular marker for proliferation, the Ki67 protein is present in cells at cycle phases G1, S, G2 and M, but absent in resting (G0) cells. Ki67 antibodies can be used instead of, or in conjunction with, BrdU to label proliferating neurons.